INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1984, p. 31-34 Vol. 34, No. 1 OO20-7713/84/010031-04$02.00/0 Copyright 0 1984, International Union of Microbiological Societies Transfer of Streptococcus faecalis and Streptococcus faecium to the Genus Enterococcus norn. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb. nov. KARL H. SCHLEIFER* AND RENATE KILPPER-BALZ Lehrstuhl fur Mikrobiologie, Technische Universitat Miinchen, D-8000 Miinchen 2, Federal Republic of Germany The results of deoxyribonucleic acid-deoxyribonucleic acid and deoxyribonucleic acid-ribosomal ribonu- cleic acid hybridization studies demonstrated that Streptococcus faecalis and Streptococcus faecium are distantly related to the non-enterococcal streptococci (Streptococcus hovis and Streptococcus equinus) of serological group D and to other streptococci. On the basis of our results and those of previous studies, we propose that S. faecalis and S. faecium be transferred to the genus Enterococcus (ex Thiercelin and Jouhaud) nom. rev. as Enterococcus faecalis (Andrewes and Horder) comb. nov. and Enterococcus faecium (Orla-Jensen) comb. nov., respectively. A description of the genus Enterococcus nom. rev. and emended descriptions of E. faecalis and E. faecium are given. The streptococci belonging to serological group D can be De Ley (4) and were corrected to the value for the reference divided into two physiologically different groups. Strepto- Escherichia coli K-12 DNA. coccus faecalis and Streptococcus faecium were placed in the enterococcus division of the streptococci, whereas RESULTS AND DISCUSSION Streptococcus bovis and Streptococcus equinus were placed in the viridans division by Sherman (21). Kalina proposed (9) The DNA base compositions, serological groups, and that Streptococcus faecalis and Streptococcus faecium peptidoglycan types of the test strains are shown in Table 1. should be transferred to the genus “Enterococcus.” This The enterococci studied (Streptococcus faecalis, Strepto- distinction between the enterococci and Streptococcus bo- coccus faecium, “Streptococcus avium” , “Streptococcus vis, Streptococcus equinus, and other streptococci has also durans”, and “Streptococcus casselifiavus”) had DNA been demonstrated by comparative biochemical (25) and G+C contents in the range from 37 to 43 mol%. These data immunological (14, 15) studies. More recently, nucleic acid are in good agreement with the data given recently by studies (7, 12) have confirmed that Streptococcus faecalis Farrow et al. (7); however, they are about 1to 2 mol% lower and Streptococcus faecium are only distantly related to than the values reported by Kilpper-Balz et al. (12), who Streptococcus bovis and Streptococcus equinus. In particu- used a value for the base composition of the reference DNA lar, deoxyribonucleic acid (DNA)-ribosomal ribonucleic acid that was too high (53 instead of 51.7 mol%). (rRNA) homology studies (12) and comparative oligonucleo- Streptococcus faecalis and its subspecies possess the Lys- tide cataloging of 16s rRNA (E. Seewaldt, Ph.D. thesis, Alaz-3peptidoglycan type and differ in this respect from all of Technische Universitat Munchen, Munich, Federal Repub- the other enterococci, which contain the LYS-D-ASPpeptido- lic of Germany, 1982) have indicated that the enterococcal glycan type (8, 12, 20). The results of previous studies, in and non-enterococcal group D streptococci belong to differ- particular those of Kilpper-Balz et al. (12) and Seewaldt ent genera. In this paper we extend these studies, and on the (Ph.D. thesis), clearly indicated that the enterococci are not basis of the data we propose that Streptococcus faecalis and closely related to the other streptococci. In this study 3H- Streptococcus faecium be transferred to the genus Entero- labeled 23s rRNAs from Streptococcus faecalis DSM 20376 coccus (ex Thiercelin and Jouhaud) nom. rev. (22). and “Streptococcus avium” DSM 20063 were hybridized with filter-bound DNAs from strains of Streptococcus faeca- MATERIALS AND METHODS lis, Streptococcus faecium, “Streptococcus durans”, The test strains which we used are listed in Table 1. The “Streptococcus casselifiavus”, and some other streptococci streptococci were cultivated in CASO-bouillon medium (E. (Table 2). Our results indicate that all of the enterococci Merck AG, Darmstadt, Germany) without aeration at 34°C. examined share high rRNA homology and are only remotely Staphylococcus sciuri and Escherichia coli were grown in related to other streptococci (Table 2). shake flasks containing glucose-peptone-yeast extract broth The results of our DNA-DNA hybridization studies (Fig. (13) at 34°C. All strains were harvested in the exponential 1) confirm the separate species status of Streptococcus phase. The procedures used to prepare cell walls and deter- faecalis and Streptococcus faecium (7, 12). All strains of mine the peptidoglycan types have been described previous- Streptococcus faecalis were closely related (DNA homology ly (19, 20). Isolation of DNA and rRNA and nucleic acid values, 88%) regardless of their subspecies status. The hybridization experiments were carried out as described Streptococcus faecium strains showed low but still signifi- previously (11-13). The stability of DNA-rRNA hybrids is cant levels of DNA homology (ca. 18%) with Streptococcus expressed by their melting temperatures. DNA base compo- faecalis. The very low levels of DNA homology (<9%) sitions were determined by thermal denaturation (16), using between Streptococcus faecalis and Streptococcus faecium a Gilford model 2600 spectrophotometer. DNA from Esche- strains and other streptococci are consistent with the DNA- richia coli K-12 was used as the standard. The guanine-plus- rRNA hybridization data (12). Three strains of Streptococ- cusfaecium (40%) cytosine (G+C) contents were calculated by the method of shared a lower level of DNA homology with the type strain of Streptococcus faecium (Fig. 1) and * Corresponding author. may represent a distinct species or at least a subspecies. The 31 32 SCHLEIFER AND KILPPER-BALZ INT.J. SYST.BACTERIOL. TABLE 1. Bacterial strains used and their serological groups, alter the end products of carbohydrate metabolism. Hydro- DNA base contents, and peptidoglycan types gen peroxide may or may not accumulate in the presence of Lance- G+C Type of oxygen. Do not contain heme compounds. Benzidine nega- Strain field content peptido- tive and usually catalase negative, but some strains may group (mol%) glycan produce pseudocatalase. Some strains synthesize cyto- “Streptococcus avium” DSM 20063 D+Q 39.2 LYS-D-ASP chromes or catalase or both when they are provided with “Streptococcus bovis” DSM 20480Ta D 38.0 Lys-Thr- hemin. The minimal nutritional requirements are generally Ala(Ser) complex. React with group D antisera; some strains also “Streptococcus casseliflavus” D 43.7 LYS-D-ASP react with group Q antisera. CCM 2478 Some strains possess respiratory quinones (menaquinones “Streptococcus casselijlavus’ ’ D 44.6 LYS-D-ASP or demethylmenaquinones). Long-chain fatty acids are pre- CCM 2479 “Streptococcus durans” CCM 5612 D 39.0 LYS-D-ASP dominantly of the straight-chain saturated or monounsatur- “Streptococcus durans” Kiel 27382 D 38.2 L~s-D-As~ ated types; some strains produce cyclopropane ring acids. “Streptococcus faecalis” DSM D 38.6 Ly~-Ala~_~ Peptidoglycan type: L~s-D-As~or Lys-Ala2-3. 20478T The G+C content of the DNA ranges from 37 to 45 mol%. “Streptococcus faecalis subsp. fae- D 37.7 Ly~-Ala~-~ Type species: Enterococcus fueculis. calis” DSM 20376 Nucleic acid hybridization studies, in particular DNA- “Streptococcusfaecalis subsp. fae- D 37.7 ‘Lys-Alat3 rRNA hybridization studies, demonstrate that members of calis” Kiel 7067 the genus Enterococcus are closely related to each other but “Streptococcus faecalis subsp. li- D 37.5 Lys-Alat3 not to members of the genus Streptococcus. Enterococci can quefaciens” Kiel 26506 “Streptococcus faecalis subsp. zy- D 38.1 L y s-Ala2-3 easily be differentiated from streptococci by their ability to mogenes’ ’ Kiel 20225 Streptococcus faecium DSM 20477T D 39.0 LYS-D-ASP Streptococcus faecium DSM 20160 D 37.4 LYS-D-ASP TABLE 2. Hybridization between ’H-labeled 23s rRNAs from Streptococcus faecium CCM 2123 D 38.6 LYS-D-ASP Streptococcus faecalis DSM 20376 and “Streptococcus uvium” Streptococcus faecium CCM 2308 D 38.1 LYS-D-ASP DSM 20063 and filter-bound DNAs from different streptococci and Streptococcus faecium CCM 2423 D 38.0 LYS-D-ASP other bacteria Streptococcus faecium CCM 2424 D 37.5 LYS-D-ASP Melting temp (“C) after Streptococcus faecium Kiel 26352 D 38.3 LYS-D-ASP hybridization with ‘H-labeled Streptococcus lactis DSM 20481T N 36.0 LYS-D-ASP rRNA from: Streptococcus mutans ATCC 25175T 37.5 L y s-Ala2.3 Source of DNA ________ Streptococcus species Kiel 9938’ L~s-D-As~ Streptococcus “SIreptococrus Q fcrecaiis aviirm” Streptococcus thermophilus DSM 38.3 Ly s-Ala2.3 DSM 20376 DSM 20063 20479 Staphylococcus sciuri ATCC 29062= 33.6 Ly s-Ala- “Streptococcus faecalis subsp. fae- 80.0 N D“ GlY4-5 calis” DSM 20376 Escherichia coli DSM 30083 51.7 m-A,pm- “Streptococcus faecalis” DSM 79.8 75.1 directc 20478 “Streptococcus faecalis subsp. zy- 79.2 ND a T = Type strain. mogenes” Kiel 20225 This strain is closely related to “Streptococcus avium” (M. D. “Streptococcus faecalis subsp. fae- 79.1 ND Collins et al., Int. J. Syst. Bacteriol., in press). calis” Kiel 7067 m-Azpm, rneso-Diaminopinelic acid. “Streptococcus faecalis subsp. li- 78.0 ND quefaciens” Kiel 26506 Streptococcus